The ability of cells to regulate the localization of molecules in both time and space is a hallmark of cellular organization and signal transduction. In the case of polarized cell migration, phosphatidylinositol (PtdIns) lipids generated by kinases and phosphatases at the plasma membrane form self-organized gradients of lipids independent of the underlying actin cytoskeleton. Although many of the proteins that regulate PtdIns lipid synthesis in vivo have been identified, the molecular basis for rapid generation and propagation of lipid gradients across intracellular membranes remains unclear. Using the design principles revealed from computational modeling of two-component signaling networks, I plan to reconstitute polarized PI(4)P and PI(4,5)P2 lipid synthesis on membranes in vitro using a synthetic biology approach. To achieve this goal, I have designed a series of chimeric lipid kinases and phosphatases that have intrinsic positive feedback built into their catalytic activity. Combining these enzymatic activities results in mutual cross-negative inhibition between both enzymes and is hypothesized to support spontaneous polarization on membranes in vitro. Using this reconstitution I will then determine which biochemical features that regulate the time evolution of polarization and fine-tuning of lipid domain size. After establishing a platform to characterize the localization and activity of lipid kinases and phosphatases on fluid lipid bilayer, I will test whether polarized PI(4)P and PI(4,5)P2 lipid domains can function as spatial cues to control the recruitment and activation of proteins that regulate actin nucleation at the plasma membrane. Taken together, these experiments should reveal key principles of cell polarization and provide a new class of biochemical reconstitutions that describe the spatiotemporal organization of signaling molecules that function on intracellular membranes.